16 research outputs found

    Hydrogen/Deuterium exchange mass spectrometric analysis of conformational changes accompanying the assembly of the yeast prion ure2p into protein fibrils.

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    International audienceThe Ure2 protein from baker's yeast (Saccharomyces cerevisiae) has prion properties. In vitro, at neutral pH, soluble Ure2p forms long, twisted fibrils. Two models have been proposed to account for Ure2p polymerization. The first postulates that a segment of 70 amino acid residues in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions. The knowledge of the three-dimensional structure of the fibrillar form of Ure2p is critical for understanding the molecular events leading to the polymerization of soluble Ure2p into fibrils and hence for the design of inhibitors that might have therapeutic potential as yeast prions possessing domains rich in N and Q residues, similar to huntingtin. Solvent-accessibility studies using hydrogen/deuterium exchange monitored by mass spectrometry (HXMS) can provide insights into the structure of the fibrillar form of Ure2p and characterize at the molecular level the conformational rearrangements that occur upon assembly, in particular through the identification of protected regions and their localization in the overall structure of the protein. We have analyzed the changes in Ure2p structure associated with its assembly into fibrils using HXMS. The deuterium incorporation profile along the sequence allows the identification of the regions that exhibit the most important conformational change. Our data reveal that Ure2p undergoes minor structural changes upon assembly. While polypeptides [82-92] and [13-37] exhibit significant increased and decreased exposure to the solvent, respectively, no marked change was observed for the rest of the protein upon assembly. Our results afford new insights into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] element in yeast

    Systemic growth of Leptosphaeria maculans from cotyledons to hypocotyls in oilseed rape: influence of number of infection sites, competitive growth and host polygenic resistance

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    International audienceThe influence of competitive effects between two isolates, of the number of infection sites on cotyledons and of host polygenic resistance on the systemic growth of Leptosphaeria maculans, the cause of phoma stem canker in oilseed rape (Brassica napus), were investigated. Controlled-condition experiments were conducted with two oilseed rape doubled haploid lines, one susceptible and the other with a high level of polygenic resistance, inoculated via wounded cotyledons with conidial suspensions obtained from two isolates. Expression of cankers in plants was enhanced by exposing inoculated plants to low temperature (6 degrees C) followed by warm temperature (20 degrees C). The fungus was detected by PCR amplifications of three minisatellite markers in all stems with visible canker symptoms and also in the stems of 14 of the 59 plants without visible cankers on the hypocotyls. Disease severity increased with the number of infection sites on cotyledons: in one of the three replicate experiments, the mean external necrosis length on the hypocotyl ranged from 6.47 to 35.3 mm for one and eight infections sites on cotyledons, respectively. The probability of an isolate reaching the hypocotyl from inoculated cotyledons decreased with increasing competing inoculum load on cotyledons: for instance, for isolate A290v it decreased from 1 when inoculated alone to 0.28 when coinoculated with six drops of competing isolate P27d. Polygenic resistance significantly reduced disease incidence and severity. For instance, in one of the three replicate experiments, disease incidence ranged from more than 74% in susceptible plants to 16% in resistant ones, while mean external necrosis length was up to 35.3 and 6.5 mm on susceptible and on resistant plants, respectively. This study offers new possibilities for assessing levels of polygenic resistance to stem canker in B. napus and studying the aggressiveness of L. maculans isolate

    Leaf Eh and pH: A Novel Indicator of Plant Stress. Spatial, Temporal and Genotypic Variability in Rice (Oryza sativa L.)

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    A wealth of knowledge has been published in the last decade on redox regulations in plants. However, these works remained largely at cellular and organelle levels. Simple indicators of oxidative stress at the plant level are still missing. We developed a method for direct measurement of leaf Eh and pH, which revealed spatial, temporal, and genotypic variations in rice. Eh (redox potential) and Eh@pH7 (redox potential corrected to pH 7) of the last fully expanded leaf decreased after sunrise. Leaf Eh was high in the youngest leaf and in the oldest leaves, and minimum for the last fully expanded leaf. Leaf pH decreased from youngest to oldest leaves. The same gradients in Eh-pH were measured for various varieties, hydric conditions, and cropping seasons. Rice varieties differed in Eh, pH, and/or Eh@pH7. Leaf Eh increases and leaf pH decreases with plant age. These patterns and dynamics in leaf Eh-pH are in accordance with the pattern and dynamics of disease infections. Leaf Eh-pH can bring new insight on redox processes at plant level and is proposed as a novel indicator of plant stress/health. It could be used by agronomists, breeders, and pathologists to accelerate the development of crop cultivation methods leading to agroecological crop protection

    Soil and plant health in relation to dynamic sustainment of Eh and pH homeostasis: A review

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    International audienceBackground: Plants perform in a specific Eh–pH spectrum and they rely on various processes to ensure their homeostasis, which plays a central role in their defense. The effects of multiple stresses, all translated into oxidative stress into the plant, and the capacity of the latter to respond to these stresses results in specific Eh–pH states in plants.Scope: We reviewed plant-invertebrate pests and plant-pathogens interactions under a Eh–pH homeostasis perspective by extensively analyzing the literature, which converges and supports a set of hypotheses. We report examples showing how the development and attacks of pests are correlated to spatio-temporal variations of Eh–pH in plants. We provide evidence-based discussionon how Eh–pH homeostasis can open a new perspective on plant health, and help unravel and disentangle the many Genotype x Environment x Management x Pest and Pathogen interactions. We propose an original perspective on energy allocation and growth-defense tradeoff by plants based on the Eh–pH homeostasis model. Finally, we show how Eh–pH conditions in the rhizosphere are the results of multiple interactions between the root system and microorganisms. Based on this, we hypothesize that soil suppressiveness is derived from soil structure leading to diverse Eh–pH niches that harbor a diversity of microorganisms.Conclusions: The Eh–pH homeostasis model proposed herein is central to soil and plant health. An Eh–pH perspective could become a very powerful tool to develop a “one health approach” unifying a large range of biophysical processes in a very coherent and consistent manner

    Gemtuzumab ozogamicin for de novo acute myeloid leukemia: final efficacy and safety updates from the open-label, phase III ALFA-0701 trial.

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    International audienceThe randomized, phase III ALFA-0701 trial showed that a reduced and fractionated dose of gemtuzumab ozogamicin added to standard front-line chemotherapy significantly improves event-free survival (EFS) in adults with de novo acute myeloid leukemia (AML). Here we report an independent review of EFS, final overall survival (OS), and additional safety results from ALFA-0701. Patients (n=271) aged 50-70 years with de novo AML were randomized to receive conventional front-line induction chemotherapy (3+7daunorubicin+cytarabine) with/without gemtuzumab ozogamicin 3 mg/m2 on days 1, 4, and 7 during induction. Patients in remission following induction therapy received 2 courses of consolidation therapy (daunorubicin+cytarabine) with/without gemtuzumab ozogamicin (3 mg/m2/day on day 1) according to their initial randomization. The primary end point was investigator-assessed EFS. Secondary end points included OS and safety. A blinded independent review confirmed the investigator-assessed EFS results [August 1, 2011; hazard ratio (HR) 0.66; 95% Confidence Interval (CI): 0.49-0.89; 2-sided P=0.006], corresponding to a 34% reduction in risk of events in the gemtuzumab ozogamicin versus control arm. Final OS at April 30, 2013 favored gemtuzumab ozogamicin but was not significant. No differences in early death rate were observed between arms. The main toxicity associated with gemtuzumab ozogamicin was prolonged thrombocytopenia. Veno-occlusive disease (including after transplant) was observed in 6 patients in the gemtuzumab ozogamicin arm and 2 in the control arm. In conclusion, gemtuzumab ozogamicin added to standard intensive chemotherapy has a favorable benefit/risk ratio. These results expand front-line treatment options for adult patients with previously untreated AML
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